Human and animal blood comprises many proteins and enzymes, which possess therapeutic and potentially life-saving properties. Some of these proteins may be found in the red blood cells whereas others are found in solution in plasma or serum. Since the middle of the 20th century such proteins have been the target for large-scale and specific isolation with the aim of purifying and standardising the proteins for use as human therapeutic agents. Examples of prominent blood proteins that are currently available as isolated therapeutic products are: albumin, immunoglobulin G, Factor VIII and alpha-1-proteinase inhibitor. Some of these proteins are produced in the scale of several thousand kg per year (albumin and IgG) while others are produced only in the gram to kilogram per year scale. However, on a worldwide basis many million liters of blood per year are processed for the purpose of isolating these proteins.
Blood, blood plasma and blood serum are extremely complicated protein containing solutions that comprises many other types of compounds other than the protein(s) or enzyme(s) of interest, all carefully balanced and regulated to work in the blood-stream in a very broad range of biochemically complicated functions such as the oxygen transport, the immuno defence and the coagulation system preventing excessive bleeding from wounds. Especially when blood is drawn from an animal and exposed to the atmosphere and the surface of different types of containers it becomes highly unstable. Although chemical agents, such as heparin and sodium citrate, can be added to increase the stability and to a certain degree prevent coagulation of the blood plasma obtained by separating the blood cells, the plasma will still be a very fragile, highly concentrated and viscous protein solution also comprising significant amounts of lipids. Despite the addition of stabilisers any handling or alteration of the plasma composition involves the risk of accidental destabilisation, which may cause activation of the coagulation cascade, precipitation of e.g. lipid components as well as denaturation of the target protein(s) and thereby makes the blood very difficult to work with. Thus, any method employed to isolate proteins from blood or blood derived solutions must take the inherent instability of the solution and the proteins themselves into consideration. This has proven to be a very significant challenge for the large-scale production of therapeutic products from blood.
Further, from a technological point of view the complexity and instability of the blood makes the separation and isolation of blood proteins much more complicated and economically demanding than the isolation of proteins from other types of protein solution such as mammalian cell culture supernatants and fermentation broth from genetically modified microorganisms as typically used in the biotech industry. Also, the biotech industry will typically only isolate one specific product from a cell culture supernatant, while for economical and ethical reasons the therapeutic blood fractionation industry generally must isolate as many products as possible from the limited amount of blood available.
The cost of blood, serum and plasma has increased very significantly during the last decades the main reason being due to increased cost of the safety measures needed to prevent viral diseases to spread from blood donors to recipients of the blood products. For more than 10-20 years the very high cost of the blood plasma as well as increased costs of implementing viral elimination steps and other safety measures during processing has put the blood fractionation industry under a significant pressure to increase the per-liter-yield of individual products such as immunoglobulin G (IgG) and alpha-1-proteinase inhibitor.
Furthermore, there is generally a strong need for expanding the number of products that can be produced from the same amount of plasma i.e. to produce an increased number of different proteins from the plasma, while still being able to produce the existing products at acceptable yields. The blood fractionation industry has experienced that these long felt needs are difficult to satisfy with known technology and although attempts have been made for a long time to employ modern adsorption techniques as an alternative to the established precipitation methods there are still significant problems in terms of economical feasibility and processing robustness of hitherto described adsorption methods.
One of the conventionally used methods for the fractionation of blood plasma or blood serum protein(s) has been described in U.S. Pat. No. 2,390,074 (Cohn et al.) which discloses a method for the fractionation of plasma or serum proteins in large-scale which utilise ethanol precipitation and regulates temperature, pH, ionic strength and time to control precipitation of certain proteins from human plasma. The fractionation method involves the stepwise addition of ethanol to the plasma raw material in order to obtain several precipitates (fractions) and corresponding supernatants comprising different enriched protein solutions.
One drawback of the ethanol precipitation method disclosed by Cohn et al. is that some proteins tend to denature during the process resulting in decreased yield of the protein to be isolated and contamination with aggregates that needs to be removed before an acceptable therapeutic product can be obtained. Furthermore, during this fractionation method precipitated proteins have to be resolubilised for further processing. Such resolubilised protein solutions may comprise significant levels of insoluble (denatured) protein and lipid material that makes it difficult and time consuming to work-up the target product, which also contributes significantly to the loss of valuable product. Additionally it is characteristic of this process that a specific protein may distribute into several of the fractions obtained during the stepwise addition of ethanol, which again results in low yields and time-consuming work-up of re-combined protein fractions.
In the fractionation of e.g. alpha-1-proteinase inhibitor or immunoglobulins, such as IgG, using the fractionation method described by Cohn et al. the yield alpha-1-proteinase inhibitor is as low as 10-20% and the yield of IgG is as low as 40-50%. However, since these products are much needed and as there is an undersupply of the product to satisfy the needs of patients, new methods for isolating such products are highly needed where the loss of product is reduced.
During the last decade many attempts have been made to develop a fractionation process which can provide an increased yield using a range of other techniques, including chromatography. However, drawbacks associated with known adsorption techniques such as low flow rates and low binding capacities resulting in low productivity as well as lack of robustness and difficulties in applying safe cleaning procedures have made it difficult to balance the yield and economy involved in the fractionation of the blood plasma and serum proteins. The core of the existing industrial manufacturing processes is therefore still based on the work of Cohn et al.
Presently used isolation and purification processes have shown to be inadequate and trace impurities resulting from inefficient purification processes may be able to stimulate an immune response in patients. Furthermore, purification processes that fail to separate active and inactive part of the product, as the presently used processes, can lead to a product with unpredictable efficacy and a specific activity, which varies between separate lots.
Even attempts to develop advanced adsorption techniques such as expanded bed adsorption, which were first introduced in the beginning of the 1990ties, have failed to improve the employment of adsorption techniques. Finette G. M. S. et al, Biotechnol. Prog., 1998, 14, pp 286-293, thus describes the application of an adsorbent having a mean particle diameter of 180 micron and a density of 1.79 g/ml for packed bed and expanded bed adsorption of α-1-proteinase inhibitor from Cohn fraction II+III. The authors conclude that a volumetric flow rate of 0.2 ml/min (corresponding to a linear flow rate of 0.1 cm/min or 60 cm/hour) will result in a yield of alpha-1-proteinase inhibitor of 50%. The authors further state that higher flow rates will decrease the yield as well as disturb the plug flow in the column. Such low flow rates are not economically attractive and are therefore prohibiting the use of e.g. expanded bed adsorption for the industrial fractionation of blood proteins.
Other attempts to apply expanded bed adsorption for isolation of human plasma proteins confirms the low flow rates applied with prior techniques. U.S. Pat. No. 6,617,133 thus describes the use of a Streamline SP adsorbent (Amersham Biosciences), which, according to the supplier, have a mean volume particle diameter of 200 micron and a density of 1.20 g/ml for the isolation of human serum albumin using a raw material application flow rate of 100 cm/hour. Such a low flow rate is limiting the productivity of the adsorption system and thus requires very large columns and results in high materials cost per unit human albumin produced.
Accordingly, a process for the fractionation of serum or plasma proteins which is fast, robust (i.e. being reliable during daily operation with low down time), specific and safe, and which at the same time provides an improved yield and purity of the products of interest during processing and thereby facilitates an improved and acceptable balance between yield and economy, compared to the conventionally used processes, e.g. the process described by Cohn et al, and which solves the above mentioned problems is therefore desired. Such a process is provided herein.